Extensive intratumor regional epigenetic heterogeneity in clear cell renal cell carcinoma targets kidney enhancers and is associated with poor outcome.

BACKGROUND: Clear cell renal cell cancer (ccRCC), the 8th leading cause of cancer-related death in the US, is challenging to treat due to high level intratumoral heterogeneity (ITH) and the paucity of druggable driver mutations. CcRCC is unusual for its high frequency of epigenetic regulator mutations, such as the SETD2 histone H3 lysine 36 trimethylase (H3K36me3), and low frequency of traditional cancer driver mutations. In this work, we examined epigenetic level ITH and defined its relationships with pathologic features, aspects of tumor biology, and SETD2 mutations. RESULTS: A multi-region sampling approach coupled with EPIC DNA methylation arrays was conducted on a cohort of normal kidney and ccRCC. ITH was assessed using DNA methylation (5mC) and CNV-based entropy and Euclidian distances. We found elevated 5mC heterogeneity and entropy in ccRCC relative to normal kidney. Variable CpGs are highly enriched in enhancer regions. Using intra-class correlation coefficient analysis, we identified CpGs that segregate tumor regions according to clinical phenotypes related to tumor aggressiveness. SETD2 wild-type tumors overall possess greater 5mC and copy number ITH than SETD2 mutant tumor regions, suggesting SETD2 loss contributes to a distinct epigenome. Finally, coupling our regional data with TCGA, we identified a 5mC signature that links regions within a primary tumor with metastatic potential. CONCLUSION: Taken together, our results reveal marked levels of epigenetic ITH in ccRCC that are linked to clinically relevant tumor phenotypes and could translate into novel epigenetic biomarkers.

Bone marrow-derived progenitor cells contribute to remodeling of the postpartum uterus.

Endometrial stem/progenitor cells play a role in postpartum uterine tissue regeneration, but the underlying mechanisms are poorly understood. While circulating bone marrow (BM)-derived cells (BMDCs) contribute to nonhematopoietic endometrial cells, the contribution of BMDCs to postpartum uterus remodeling is unknown. We investigated the contribution of BMDCs to the postpartum uterus using 5-fluorouracil-based nongonadotoxic BM transplant from green fluorescent protein (GFP) donors into wild-type C57BL/6J female mice. Flow cytometry showed an influx of GFP+ cells to the uterus immediately postpartum accounting for 28.7% of total uterine cells, followed by a rapid decrease to prepregnancy levels. The majority of uterine GFP+ cells were CD45+ leukocytes, and the proportion of nonhematopoietic CD45-GFP+ cells peaked on postpartum day (PPD) 1 (17.5%). Immunofluorescence colocalization of GFP with CD45 pan-leukocyte and F4/80 macrophage markers corroborated these findings. GFP+ cells were found mostly in subepithelial stromal location. Importantly, GFP+ cytokeratin-positive epithelial cells were found within the luminal epithelium exclusively on PPD1, demonstrating direct contribution to postpartum re-epithelialization. A subset (3.2%) of GFP+ cells were CD31+CD45- endothelial cells, and found integrated within blood vessel endothelium. Notably, BM-derived GFP+ cells demonstrated preferential proliferation (PCNA+) and apoptosis (TUNEL+) on PPD1 vs resident GFP- cells, suggesting an active role for BMDCs in rapid tissue turnover. Moreover, GFP+ cells gradually acquired cell senescence together with decreased proliferation throughout the postpartum. In conclusion, BM-derived progenitors were found to have a novel nonhematopoietic cellular contribution to postpartum uterus remodeling. This contribution may have an important functional role in physiological as well as pathological postpartum endometrial regeneration.

CXCR4 or CXCR7 antagonists treat endometriosis by reducing bone marrow cell trafficking.

Adult stem cells have a major role in endometrial physiology, including remodelling and repair. However, they also have a critical role in the development and progression of endometriosis. Bone marrow-derived stem cells engraft eutopic endometrium and endometriotic lesions, differentiating to both stromal and epithelial cell fates. Using a mouse bone marrow transplantation model, we show that bone marrow-derived cells engrafting endometriosis express CXCR4 and CXCR7. Targeting either receptor by the administration of small molecule receptor antagonists AMD3100 or CCX771, respectively, reduced BM-derived stem cell recruitment into endometriosis implants. Endometriosis lesion size was decreased compared to vehicle controls after treatment with each antagonist in both an early growth and established lesion treatment model. Endometriosis lesion size was not effected when the local effects of CXCL12 were abrogated using uterine-specific CXCL12 null mice, suggesting an effect primarily on bone marrow cell migration rather than a direct endometrial effect. Antagonist treatment also decreased hallmarks of endometriosis physiopathology such as pro-inflammatory cytokine production and vascularization. CXCR4 and CXCR7 antagonists are potential novel, non-hormonal therapies for endometriosis.

Adult bone marrow progenitors become decidual cells and contribute to embryo implantation and pregnancy.

Decidua is a transient uterine tissue shared by mammals with hemochorial placenta and is essential for pregnancy. The decidua is infiltrated by many immune cells promoting pregnancy. Adult bone marrow (BM)-derived cells (BMDCs) differentiate into rare populations of nonhematopoietic endometrial cells in the uterus. However, whether adult BMDCs become nonhematopoietic decidual cells and contribute functionally to pregnancy is unknown. Here, we show that pregnancy mobilizes mesenchymal stem cells (MSCs) to the circulation and that pregnancy induces considerable adult BMDCs recruitment to decidua, where some differentiate into nonhematopoietic prolactin-expressing decidual cells. To explore the functional importance of nonhematopoietic BMDCs to pregnancy, we used Homeobox a11 (Hoxa11)-deficient mice, having endometrial stromal-specific defects precluding decidualization and successful pregnancy. Hoxa11 expression in BM is restricted to nonhematopoietic cells. BM transplant (BMT) from wild-type (WT) to Hoxa11-/- mice results in stromal expansion, gland formation, and marked decidualization otherwise absent in Hoxa11-/- mice. Moreover, in Hoxa11+/- mice, which have increased pregnancy losses, BMT from WT donors leads to normalized uterine expression of numerous decidualization-related genes and rescue of pregnancy loss. Collectively, these findings reveal that adult BMDCs have a previously unrecognized nonhematopoietic physiologic contribution to decidual stroma, thereby playing important roles in decidualization and pregnancy.

Bone-marrow-derived endothelial progenitor cells contribute to vasculogenesis of pregnant mouse uterus†.

Angiogenesis is essential for cyclic endometrial growth, implantation, and pregnancy maintenance. Vasculogenesis, the formation of new blood vessels by bone marrow (BM)-derived endothelial progenitor cells (EPCs), has been shown to contribute to endometrial vasculature. However, it is unknown whether vasculogenesis occurs in neovascularization of the decidua during pregnancy. To investigate the contribution of BM-derived EPCs to vascularization of the pregnant uterus, we induced non-gonadotoxic submyeloablation by 5-fluorouracil administration to wild-type FVB/N female mice recipients followed by BM transplantation from transgenic mice expressing green fluorescent protein (GFP) under regulation of Tie2 endothelial-specific promoter. Following 1 month, Tie2-GFP BM-transplanted mice were bred and sacrificed at various gestational days (ED6.5, ED10.5, ED13.5, ED18.5, and postpartum). Bone-marrow-transplanted non-pregnant and saline-injected pregnant mice served as controls (n = 5-6/group). Implantation sites were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence. While no GFP-positive EPCs were found in non-pregnant or early pregnant uteri of BM-transplanted mice, GFP-positive EPCs were first detected in pregnant uterus on ED10.5 (0.12%) and increased as the pregnancy progressed (1.14% on ED13.5), peaking on ED18.5 (1.42%) followed by decrease in the postpartum (0.9%). The percentage of endothelial cells that were BM-derived out of the total endothelial cell population in the implantation sites (GFP+CD31+/CD31+) were 9.3%, 15.8%, and 6.1% on ED13.5, ED18.5, and postpartum, respectively. Immunohistochemistry demonstrated that EPCs incorporated into decidual vasculature, and immunofluorescence showed that GFP-positive EPCs colocalized with CD31 in vascular endothelium of uterine implantation sites, confirming their endothelial lineage. Our findings indicate that BM-derived EPCs contribute to vasculogenesis of the pregnant mouse decidua.

A snapshot of gut microbiota of an adult urban population from Western region of India.

The human gut microbiome contributes to a broad range of biochemical and metabolic functions that directly or indirectly affect human physiology. Several recent studies have indicated that factors like age, geographical location, genetic makeup, and individual health status significantly influence the diversity, stability, and resilience of the gut microbiome. Of the mentioned factors, geographical location (and related dietary/socio-economic context) appears to explain a significant portion of microbiome variation observed in various previously conducted base-line studies on human gut microbiome. Given this context, we have undertaken a microbiome study with the objective of cataloguing the taxonomic diversity of gut microbiomes sampled from an urban cohort from Ahmedabad city in Western India. Computational analysis of microbiome sequence data corresponding to 160 stool samples (collected from 80 healthy individuals at two time-points, 60 days apart) has indicated a Prevotella-dominated microbial community. Given that the typical diet of participants included carbohydrate and fibre-rich components (predominantly whole grains and legume-based preparations), results appear to validate the proposed correlation between diet/geography and microbiome composition. Comparative analysis of obtained gut microbiome profiles with previously published microbiome profiles from US, China, Finland, and Japan additionally reveals a distinct taxonomic and (inferred) functional niche for the sampled microbiomes.

Influence of tick and mammalian physiological temperatures on Borrelia burgdorferi biofilms.

The spirochaete bacterium Borrelia burgdorferisensu lato is the aetiologic agent of Lyme disease. Borrelia is transmitted to mammals through tick bite and is adapted to survive at tick and mammalian physiological temperatures. We have previously shown that B. burgdorferi can exist in different morphological forms, including the antibiotic-resistant biofilm form, in vitro and in vivo. B. burgdorferi forms aggregates in ticks as well as in humans, indicating potential of biofilm formation at both 23 and 37 °C. However, the role of various environmental factors that influence Borrelia biofilm formation remains unknown. In this study, we investigated the effect of tick (23 °C), mammalian physiological (37 °C) and standard in vitro culture (33 °C) temperatures with the objective of elucidating the effect of temperature on Borrelia biofilm phenotypes invitro using two B. burgdorferisensu stricto strains (B31 and 297). Our findings show increased biofilm quantity, biofilm size, exopolysaccharide content and enhanced adherence as well as reduced free spirochaetes at 37 °C for both strains, when compared to growth at 23 and 33 °C. There were no significant variations in the biofilm nano-topography and the type of extracellular polymeric substance in Borrelia biofilms formed at all three temperatures. Significant variations in extracellular DNA content were observed in the biofilms of both strains cultured at the three temperatures. Our results indicate that temperature is an important regulator of Borrelia biofilm development, and that the mammalian physiological temperature favours increased biofilm formation in vitro compared to tick physiological temperature and in vitro culture temperature.

A Murine 5-Fluorouracil-Based Submyeloablation Model for the Study of Bone Marrow-Derived Cell Trafficking in Reproduction.

Bone marrow (BM)-derived cells (BMDCs) contribute to endometrial regeneration. Our objective was to develop a nongonadotoxic mouse BM transplant (BMT) model using 5-fluorouracil (5-FU) for investigating BMDCs trafficking in reproduction. Female C57BL/6J mice received either single (CTX-1) or paired (CTX-2) 5-FU (150 mg/kg) dose, or single (CTX-1+SCF) or paired-dose (CTX-3+SCF) 5-FU with stem cell factor (SCF). Control mice received BMT only or saline. BM cells (20 × 106) from transgenic green-fluorescent protein (GFP) mice were injected iv. For fertility experiment, mice were mated on day 28 after BMT. Alternatively, mice were killed 1 month after BMT and BMDCs recruitment to the uterus was determined. Mice receiving 5-FU ± SCF showed intact ovarian function and fertility. CTX-3+SCF resulted in greatest BM donor chimerism at 1 month (∼45%). Flow cytometry analysis demonstrated that 6.6% of total uterine cells in CTX-3+SCF mice were GFP+ BMDCs. Remarkably, this was about 40- and 80-fold greater than BMDCs in uterus of CTX-1 or BMT only mice (6.6% vs 0.16% vs 0.08%, respectively, P < .001). Immunohistochemical analysis showed that BMDCs in the uterus were mostly localized to the endometrial stroma (71.8%). The majority of endometrial BMDCs colocalized with the pan-leuokocyte CD45 marker (58.5%), but 41.5% were CD45-negative. Cytokeratin and CD31 staining showed that the GFP+CD45- cells were not epithelial or endothelial, confirming their stromal identity. We demonstrate that paired-dose 5-FU regimen results in efficient BM donor chimerism while maintaining ovarian function and fertility. This model could be used for studying BMDCs trafficking to the uterus in various reproductive physiological and pathological conditions.